Preparation method and application of novel crown tandem epitope peptide virus-like particle vaccine

Abstract

The invention relates to the technical field of vaccine production, and discloses a preparation method of a novel crown tandem epitope peptide virus-like particle vaccine, the novel crown tandem epitope peptide is formed by sequentially and linearly connecting an epitope peptide S450-500, an epitope peptide S556-589, an epitope peptide S624-690, an epitope peptide S809-835 and an epitope peptide S1140-1166 in series, the preparation method of the novel crown tandem epitope peptide virus-like particle vaccine comprises the following steps: inserting the novel crown tandem epitope peptide into an immunodominant epitope of HBc by using a flexible linker 3, and purifying through an expression system, and the invention also discloses an application of the novel crown tandem epitope peptide virus-like particle vaccine in preparation of the novel crown tandem epitope peptide virus-like particle vaccine. The novel crown tandem epitope peptide virus-like particle vaccine prepared by the invention can cope with continuous variation of the SARS-CoV-2 virus.

Description

Technical Field

[0001] The present invention relates to the field of vaccine production technology, and specifically to a preparation method and application of a novel coronavirus tandem epitope peptide virus-like particle vaccine. Background Art

[0002] SARS-CoV-2 is an RNA virus that can bind to the host cell's ACE2 to induce conformational changes in the S1 subunit and expose the S2' cleavage site in the S2 subunit. Cleavage of the S2' site exposes the fusion peptide (FP), and the dissociation of S1 from S2 causes drastic conformational changes in the S2 subunit, especially in the heptad repeat sequence, pushing the fusion peptide forward into the target membrane and initiating membrane fusion. The fusion between the viral membrane and the cell membrane forms a fusion pore through which the viral RNA is released into the host cell cytoplasm for uncoating and replication.

[0003] Vaccines remain the most cost-effective and successful intervention for the control and prevention of the new coronavirus. Existing SARS-CoV-2 vaccines mainly include inactivated, live attenuated vaccines, viral vector vaccines, protein subunit vaccines, RNA, DNA and virus-like particles (VLP) vaccines. Emerging SARS-CoV-2 variants continue to evade the neutralization provided by current vaccines and antibodies from natural infections, and with the emergence of BQ.1, BQ.1.1 and XBB.1 lineages, all approved therapeutic antibodies have completely lost their efficacy. In addition, under the selective immune pressure of the virus's widespread prevalence in the general population, SARS-CoV-2 seems likely to continue to exist. Therefore, it is necessary to develop new antibodies to fight against currently circulating variants and prepare for future strains. In general, traditional vaccines based on attenuated or inactivated viruses show excellent immunogenicity; mainly due to several key advantages, including the replication capacity of attenuated viruses; their repetitive surface geometry; particle properties and the ability to stimulate innate and adaptive immune responses. Virus-like particles (VLPs) are multi-protein supramolecular structures that carry many viral properties, and therefore, VLP-based vaccines exhibit most of the advantages of traditional vaccines. But the lack of a viral genome precludes their ability to replicate, making them safe templates for vaccine development.

[0004] The core protein (HBc) of hepatitis B virus (HBV) is a 21 kDa polypeptide that spontaneously assembles into an icosahedral particle structure with a diameter of approximately 36 nm, consisting of 120 homodimers. The core protein contains a potent helper T epitope in its 1-149 aa sequence. These two factors make HBc an exceptionally strong immunogen and an attractive candidate as a carrier of foreign epitopes. The immunodominant epitope c / e1 (79-81 aa) of HBc is believed to be a superior position to convey high immunogenicity to heterologous sequences, and the inserted sequence is more immunogenic than in the context of its native protein. These properties of HBc make it very suitable as a VLP vaccine vector.

[0005] Therefore, in order to cope with the continuous mutation of SARS-CoV-2 virus and develop a safe and effective SARS-CoV-2 vaccine, the present invention designs a method for preparing a novel coronavirus tandem epitope peptide virus-like particle vaccine. That is, multiple epitope peptides with relatively conservative sequences on the novel coronavirus S protein that can induce neutralization reactions and produce antibodies are selected and connected with flexible linkers and inserted into the immunodominant region of HBc, and then self-assembled into virus-like particles through multiple expression systems such as Escherichia coli, mammalian cells, and yeast. Summary of the invention

[0006] The purpose of the present invention is to provide a technical method for selecting multiple epitope peptides with relatively conservative sequences on the S protein of the new coronavirus and capable of inducing a neutralizing reaction to produce antibodies, connecting them with a flexible linker and inserting them into the immunodominant region of HBc, and self-assembling them into virus-like particles through various expression systems such as Escherichia coli, mammalian cells, and yeast, so as to cope with the technical problem of the continuous mutation of the SARS-CoV-2 virus.

[0007] In order to achieve the above object, the present invention is achieved in the following ways:

[0008] The present invention proposes a novel coronavirus tandem epitope peptide, which is composed of a core region epitope peptide S450-500 that binds to ACE2 in the RBD domain, an epitope peptide S556-589 that can induce the production of neutralizing antibodies near the RBD domain and hinder the binding of the RBD domain and ACE2 through steric hindrance, an epitope peptide S624-690 located at the C-terminus of the S1 subunit, which can induce antibodies to prevent membrane fusion by inhibiting host protease-mediated protein hydrolysis, an epitope peptide S809-835 located at the C-terminus of the S2 subunit fusion peptide, which can target and induce immune responses of CD4+T cells and CD8+T cells, and an epitope peptide S1140-1166 located at the C-terminus of the spike protein, which is relatively conserved in coronaviruses, which are linearly connected in sequence.

[0009] Preferably, the amino acid sequence of the epitope peptide S450-500 is shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the epitope peptide S556-589 is shown in SEQ ID NO: 4;

[0011] The amino acid sequence of the epitope peptide S624-690 is shown in SEQ ID NO: 5;

[0012] The amino acid sequence of the epitope peptide S809-835 is shown in SEQ ID NO: 6;

[0013] The amino acid sequence of the epitope peptide S1140-1166 is shown in SEQ ID NO: 7;

[0014] There is a spacer sequence between any two adjacent epitope peptide segments, and the spacer sequence is GGGGS.

[0015] The present invention also provides a novel coronavirus tandem epitope peptide virus-like particle vaccine, comprising the core protein of hepatitis B virus and the above-mentioned novel coronavirus tandem epitope peptide.

[0016] Preferably, there is a spacer sequence between the core protein of the hepatitis B virus and the new coronavirus tandem epitope peptide; the spacer sequence is GGGGS.

[0017] Preferably, the amino acid sequence of the hepatitis B virus core protein and the new coronavirus tandem epitope peptide fusion gene is as shown in SEQ ID NO: 1.

[0018] The present invention also provides a nucleic acid encoding the active ingredient of the above-mentioned new coronavirus tandem epitope peptide virus-like particle vaccine, and the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 2.

[0019] The present invention also provides a method for preparing a new coronavirus tandem epitope peptide virus-like particle vaccine, which comprises: using a flexible linker 3 to insert the new coronavirus tandem epitope peptide of any one of claims 1-2 into the immunodominant epitope of HBc, i.e., between positions 79-81 of the amino acid sequence, to obtain a recombinant plasmid, and then purifying the recombinant plasmid through an expression system to obtain the new coronavirus tandem epitope peptide virus-like particle vaccine.

[0020] Preferably, the expression system is any one of a yeast expression system and a mammalian expression system.

[0021] The present invention also provides a novel coronavirus tandem epitope peptide virus-like particle vaccine prepared by the preparation method of the novel coronavirus tandem epitope peptide virus-like particle vaccine.

[0022] The present invention also provides the use of the above-mentioned new coronavirus tandem epitope peptide virus-like particle vaccine in the preparation of the new coronavirus tandem epitope peptide virus-like particle vaccine.

[0023] The following are the sequences involved in the solution of the present invention:

[0024] SEQ ID NO: 1

[0025] MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCS

[0026] PHHTALRQAILCWGELMNLATWVGSNLEDPGGGGSNYLYRLFRKSNLK

[0027] PFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPGGGGSNKKFLPFQ

[0028] QFGRDIADTTDAVRDPQTLEILDITPGGGGSIHADQLTPTWRVYSTGSNV

[0029] FQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQGG

[0030] GGSPSKPSKRSFIEDLLFNKVTLADAGFIKGGGGSPLQPELDSFKEELDK

[0031] YFKNHTSPDVDLGGGGSSRELVVSYVNVNMGLKIRQLLWFHISCLTFGR

[0032] ETVLEYLVSFGVWIRTPPAYRPPYAPILSTLPETTVVGGGGSHHHHHH

[0033] SEQ ID NO: 2

[0034] ATGGACATCGACCCGTACAAAGAATTCGGTGCTTCTGTTGAACTG

[0035] CTGTCTTTCCTGCCGTCTGACTTCTTCCCGTCTATCCGTGACCTGCTGG

[0036] ACACCGCTTCTGCTCTGTACCGTGAAGCTCTGGAATCTCCGGAACAC

[0037] TGCTCTCCGCACCACACCGCTCTGCGTCAGGCTATCCTGTGCTGGGGT

[0038] GAACTGATGAACCTGGCTACCTGGGTTGGTTCTAACCTGGAAGACCC

[0039] GGGCGGCGGCGGCAGCAACTACCTGTACCGCCTGTTCCGCAAGAGCA

[0040] ACCTGAAGCCCTTCGAGCGCGACATCAGCACCGAGATCTACCAGGCC

[0041] GGCAACAAGCCCTGCAACGGCGTGGCCGGCTTCAACTGCTACTTCCC

[0042] CCTGCGCAGCTACGGCTTCCGCCCCGGCGGCGGCGGCAGCAACAAG

[0043] AAGTTCCTGCCCTTCCAGCAGTTCGGCCGCGACATCGCCGACACCAC

[0044] CGACGCCGTGCGCGACCCCCAGACCCTGGAGATCCTGGACATCACCC

[0045] CCGGCGGCGGCGGCAGCATCCACGCCGACCAGCTGACCCCCACCTG

[0046] GCGCGTGTACAGCACCGGCAGCAACGTGTTCCAGACCCGCGCCGGCT

[0047] GCCTGATCGGCGCCGAGCACGTGAACAACAGCTACGAGTGCGACATC

[0048] CCCATCGGCGCCGGCATCTGCGCCAGCTACCAGACCCAGACCAAGAG

[0049] CCACCGCCGCGCCCGCAGCGTGGCCAGCCAGGGCGGCGGCGGCAGC

[0050] CCCAGCAAGCCCAGCAAGCGCAGCTTCATCGAGGACCTGCTGTTCAA

[0051] CAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGGGCGGCGGCGGC

[0052] AGCCCCCTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACA

[0053] AGTACTTCAAGAACCACACCAGCCCCGACGTGGACCTGGGCGGCGG

[0054] CGGCAGCTCTCGTGAACTGGTTGTTTCTTACGTTAACGTTAACATGGG

[0055] TCTGAAAATCCGTCAGCTGCTGTGGTTCCACATCTCTTGCCTGACCTT

[0056] CGGTCGTGAAACCGTTCTGGAATACCTGGTTTCTTTCGGTGTTTGGATCCGTACCCCGCCGGCTTACCGTCCGCCGTACGCTCCGATCCTGTCTACCCTGCCGGAAACCACCGTTGTTGGCGGCGGCGGCAGCCACCACCACCACCACCAC

[0057] SEQ ID NO:3

[0058] NYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRP

[0059] SEQ ID NO:4

[0060] NKKFLPFQQFGRDIADTTDAVRDPQTLEILDITP

[0061] SEQ ID NO:5

[0062] IHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQ

[0063] SEQ ID NO:6

[0064] PSKPSKRSFIEDLLFNKVTLADAGFIK

[0065] SEQ ID NO:7

[0066] PLQPELDSFKEELDKYFKNHTSPDVDL

[0067] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0068] 1. The new coronavirus tandem epitope peptide virus-like particle vaccine provided by the present invention is highly conservative against coronaviruses and new coronavirus mutant strains, has good immunogenicity, and can stimulate the body to produce antigen-specific antibodies.

[0069] 2. The recombinant protein of the present invention can utilize the yeast expression system as an expression vector, which has the advantages of high yield, low production cost, and high protein purity, and is suitable for large-scale preparation of new crown vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a schematic diagram of the hepatitis B core protein-new crown tandem epitope peptide fusion gene in Example 2 of the present invention;

[0071] Figure 2 is the SDS-PAGE analysis of the fusion protein expressed by E. coli in Example 3 of the present invention (M: standard protein molecular weight, 1: Rosetta bacterial protein, 2: uninduced bacterial protein after transformation, 3: induced bacterial protein, 4: precipitate of bacterial lysis after induction);

[0072] Figure 3 The affinity chromatography purification and assembly identification of the fusion protein in Example 3 of the present invention (A is the SDS-PAGE analysis of the affinity chromatography purification of the fusion protein, wherein M: standard protein molecular weight, 1: Rosetta bacterial protein, 2: uninduced bacterial protein, 3: induced bacterial protein, 4: supernatant of lysed bacteria after induction, 5: column liquid of supernatant of lysed bacteria, 6-7: washing liquid, 9-10: eluent of target protein; B is a transmission electron micrograph of the fusion protein after self-assembly);

[0073] Figure 4 3 is a diagram showing the identification results of the expression of the Pichia fusion protein in Example 3 of the present invention (A is an SDS-PAGE analysis of the expression of the Pichia fusion protein, wherein M is a standard protein molecular weight, 1 is induced expression of BMMY after transformation for 24 hours, 2 is induced expression of untransformed yeast for 24 hours, 3 is induced expression of BMMY after transformation for 72 hours, 4 is induced expression of untransformed yeast for 72 hours; B is a transmission electron micrograph);

[0074] Figure 5 The purification and identification of the mammalian cell fusion protein in Example 3 of the present invention (A is the WB result after purification by sucrose density gradient centrifugation, wherein M: standard protein molecular weight, 1-11: samples of layers 1-11 after sucrose density gradient centrifugation; B is the transmission electron micrograph of the ninth layer of the sucrose gradient);

[0075] Figure 6 These are the results of body weight changes and serum-specific antibody detection of immunized mice in Example 4 of the present invention (A is a graph showing changes in mouse body weight, and B is a graph showing the results of detection of anti-recombinant fusion protein virus-like particle antibodies in mouse serum). DETAILED DESCRIPTION

[0076] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0077] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0078] Example 1

[0079] Screening for suitable novel coronavirus epitope peptides

[0080] The mutation sites of the currently popular new coronavirus variants are mainly concentrated in the RBD segment, and these mutations will reduce the protective effect of vaccines or neutralizing antibodies. Many studies have reported that antibodies triggered by other regions of the S protein other than the RBD domain are also crucial. Based on the above reasons, a variety of antigen epitope prediction software were used to predict B cell epitopes for the entire sequence of the S protein. Peptides 1 to 10 were selected according to the ranking of the prediction scores of different software. After a comprehensive comparison of the positioning, length and differences of the amino acid sequences, 11 candidate epitope peptide sequences were recombinantly designed and named according to the amino acid sequence of the S protein (S100-129, S221-234, S269-296, S371-410, S450-500, S556-589, S624-690, S809-835, 972-1004, S1140-1166, S1209-1234). Eleven candidate epitope polypeptide sequences were synthesized in vitro and terminally labeled with biotin to obtain 11 synthetic peptides.

[0081] Antigenicity identification of synthetic peptides. Eleven synthetic peptides were coated on streptavidin ELISA plates and blocked with biotin. ELISA was performed on 18 SARS convalescent sera, and the test results are shown in Table 1.

[0082] Table 1 ELISA results of 11 synthetic peptides

[0083]

[0084] The results in Table 1 show that among the 11 antigenic peptides, the antigenic peptides with a detection rate of more than 50% for 18 SARS convalescent sera correspond to epitope peptide sequences S450-500, S1140-1166, and S624-690, respectively; among them, the antigenic peptides with epitope peptide sequences S556-589 and S809-835 have a detection rate of nearly 50% for SARS convalescent sera. In other words, the five short peptides (S450-500, S556-589, S624-690, S809-835, and S1140-1166) were preliminarily identified to have a high binding rate with antibodies in convalescent sera.

[0085] Therefore, the above 5 epitope peptides with a higher binding rate with antibodies in convalescent serum were selected from the entire region of the SARS-CoV-2 Spike protein for the implementation of the present invention.

[0086] Table 2 Amino acid sequence information of 5 epitope peptides

[0087]

[0088] It should be further explained that S450-500 belongs to the receptor binding motif (RBM) in the RBD, and is the core region in the RBD domain that binds to ACE2; S556-589 is near the RBD domain, and most of this peptide segment is located on the surface of the spike protein. Many studies have shown that this site can induce neutralizing antibodies, which hinder the binding of the RBD domain and ACE2 through steric hindrance; S624-690 is located at the C-terminus of the S1 subunit, which is a hot spot for linear epitope research. The antibodies induced by this site may prevent the membrane fusion process by inhibiting host protease-mediated protein hydrolysis; S809-835 is located at the C-terminus of the S2 subunit fusion peptide (FP), which can target and induce immune responses of CD4+T cells and CD8+T cells; S1140-1166 is located at the C-terminus of the spike protein, which is relatively conserved in coronaviruses and has the potential to be designed as a broad-spectrum vaccine to cope with the continuous mutation of the virus.

[0089] Example 2

[0090] Construction of recombinant plasmid

[0091] The gene sequences of HBc and the five novel coronavirus epitope peptides selected in Example 1 were queried by NCBI, and the five epitope peptides selected in Example 1 were serially linked in the order of their appearance on the S protein using a flexible linker (GGGGS) 3 and inserted into the immunodominant epitope of HBc, i.e., between positions 79 and 81 of the amino acid sequence, to construct a recombinant gene. The amino acid sequence of the recombinant gene is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the amino acid sequence of the recombinant gene is shown in SEQ ID NO: 2. The designed recombinant sequence was sent to Qingke Biotechnology Co., Ltd. for synthesis, and the constructed recombinant protein was shown in Figure 1 As shown. Since virus-like particle (VLP) vaccines can be produced in more than 170 different expression host systems, including bacteria, insects, yeast or mammalian cells, the present invention selects three expression systems, namely Escherichia coli, Pichia pastoris and mammalian cells. Therefore, PET-32a vector, pPICZαA vector and pcDNA3.1 vector are selected to synthesize recombinant expression plasmids, and a 6×His tag is added to the C-terminus of the fusion gene to facilitate the purification of the recombinant fusion protein. Thus, three recombinant expression plasmids, namely prokaryotic recombinant plasmid, yeast recombinant plasmid and cell recombinant plasmid, are obtained respectively.

[0092] Example 3

[0093] Virus-like particle (VLP) vaccine production

[0094] 1. Production of virus-like particles (VLP) vaccines in E. coli expression system

[0095] 1.1 Expression of fusion protein in E. coli

[0096] The prokaryotic recombinant plasmid constructed in Example 2 was transformed into Rosetta competent cells, spread on LB solid medium plates (containing 100 μg / ml Amp), and cultured overnight at 37°C for 12-14 hours. The next day, a single colony was picked and placed in LB liquid medium (containing 100 μg / ml Amp) and cultured at 37°C, 220 rpm, and shaken for 6-7 hours. Then, the shaken bacterial solution was inoculated into autoinduction medium (12 g / L tryptone, 24 g / L yeast extract, 0.8% glycerol, 5 g / L lactose, 0.15 g / L glucose, 0.17 mol / L KH2PO4, 0.72 mol / L K2HPO4, 2 mmol / LMgSO4, 0.4% aspartic acid, containing 100 μg / mL Amp) at a ratio of 1:100, and cultured at 37°C and 250 rpm until the OD600 value of the bacterial solution reached 1.2, then the temperature was lowered to 25°C for overnight induction for 18 h (see the results). Figure 2 ).

[0097] Figure 2The results of SDS-PAGE analysis showed that compared with the uninduced bacterial protein, after induction, there was an obvious target protein expression band at about 43KDa, indicating that the expression of the recombinant protein was successfully induced. After ultrasonic lysis and centrifugation, the target protein could be detected in the supernatant and precipitate of the bacterial lysate.

[0098] 1.2 Purification and self-assembly of fusion protein

[0099] The overnight induced bacterial solution was centrifuged at 4000rpm for 20min, the supernatant was discarded, and the bacteria were collected. The bacteria were resuspended in a lysis solution (1.59g / LNa2CO3, 2.93g / L NaHCO3, 5mM imidazole, 1mg / ml protease inhibitor, 0.5% Triton X-100, pH 7.5) and the bacteria were broken by ultrasonication in an ice bath. The lysis liquid was centrifuged at 4°C, 12000rpm for 20min, and the supernatant and precipitate were separated. The fusion protein monomer in the lysis supernatant was purified by Ni affinity column. After the Ni affinity column was balanced with 5 column volumes of balance solution (1.59 g / L Na2CO3, 2.93 g / L NaHCO3, 5 mM imidazole, pH 7.5), the supernatant of the broken bacteria was poured into the Ni affinity column, and the column was rotated and bound on the Ferris wheel at room temperature for 2 hours, and then washed 3 times with 10 column volumes of washing solution (1.59 g / L Na2CO3, 2.93 g / L NaHCO3, 20 mM imidazole, pH 7.5) each time to remove impurities. Finally, the target protein was eluted with elution solution (1.59 g / L Na2CO3, 2.93 g / L NaHCO3, 500 mM imidazole, pH 7.5). The eluted target protein was dialyzed to gradually reduce the imidazole concentration, and the fusion protein monomers self-assembled into virus-like particles during the dialysis process. The assembled virus-like particles were identified by SDS-PAGE, Western Blot, Particle gel and transmission electron microscopy (see Figure 3 ).

[0100] Figure 3 The results showed that when the fusion protein produced by E. coli self-induction was purified by Ni column affinity chromatography, part of the target protein was found in the column liquid and the washing liquid of the bacteria lysis supernatant ( Figure 3 A), the His tag at the C-terminus was not fully exposed, and the target protein was not easy to bind to the Ni column and the binding was not strong. The purified E. coli fusion protein was dialyzed and self-assembled, and the transmission electron microscope ( Figure 3 B) It was found that the fusion protein indeed self-assembled into virus-like particles.

[0101] 2. Producing virus-like particle (VLP) vaccines in yeast expression system

[0102] 2.1 Linearization of recombinant plasmid

[0103] Sac I single enzyme digestion yeast expression plasmid linearized recombinant plasmid. 100μl enzyme digestion reaction system is as follows: Sac I enzyme 4μl, Cutsmartbuffer (10×) 10μl, yeast recombinant plasmid constructed in Example 2 10μg, enzyme-free water to 100μl, mix well and incubate in a 37℃ water bath for 4h, enzyme digestion product is detected by 1% agarose gel electrophoresis. Then the enzyme digestion product is purified by phenol chloroform extraction method. The enzyme-digested product was made up to 500 μl with enzyme-free water, and an equal volume of phenol was added. The upper aqueous phase was taken; an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added, and the upper aqueous phase was taken by centrifugation at 4°C, 12000 rpm for 10 min; an equal volume of chloroform: isoamyl alcohol (24:1) was added, and the upper aqueous phase was taken by centrifugation at 4°C, 12000 rpm for 10 min; 1 / 10 volume of 3M sodium acetate, 2.5 times volume of anhydrous ethanol and 1 μl of glycogen were added, mixed, and placed at -20°C for 1-2h, centrifuged at 4°C, 12000 rpm for 15 min, the supernatant was discarded, the precipitate was dried for 5-10 min, 50 μl of enzyme-free water was added to dissolve the precipitate, and the concentration of the linear plasmid after enzyme digestion was detected.

[0104] 2.2 Competent yeast culture and transformation

[0105] Pick a wild-type yeast colony from the cultured GS115 single colony plate and inoculate it into 10ml YPD medium (10g / L yeast extract, 20g / L peptone, 20g / L glucose, 100μg / ml chloramphenicol), and place it in a 30℃ shaker for overnight culture. Centrifuge the overnight cultured bacterial solution at 4℃, 4000rpm for 5min and discard the supernatant. Wash the bacteria, add 30ml pre-cooled sterile water to resuspend the bacteria, centrifuge at 4℃, 4000rpm for 5min, and discard the supernatant; after washing twice, add 1ml pre-cooled 1M sorbitol solution to resuspend the bacteria, and place the competent yeast on ice for later use. Electrotransform competent yeast, add 80μl competent yeast to 5μg purified linear plasmid, blow evenly and place on ice for 5min, transfer to pre-cooled electrotransformer, add 500μl 1M sorbitol to the electrotransformer immediately after electrotransformation to blow evenly, transfer to 1.5ml EP tube, place in 30℃ incubator and incubate for 2h. Take 100μl of the incubated bacterial solution and apply it on YPD plate (10g / L yeast extract, 20g / L peptone, 20g / L glucose, 2% agar powder).

[0106] 2.3 Yeast induction expression of fusion protein

[0107] Pick a single colony from the YPD plate and inoculate it into 2ml YPD medium (containing 100μg / ml chloramphenicol) and culture it in a shaking incubator overnight at 30℃. The bacterial solution after small shaking was first subjected to PCR to identify whether the plasmid was transformed into the competent yeast. The identified bacterial solution was inoculated into the expansion medium BMGY medium (10g / L yeast extract, 20g / L peptone, 0.2g / L biotin, 13.4g / L basic nitrogen source medium, 1% glycerol, 13mmol / L K2HPO4, 87mmol / L KH2PO4) at a ratio of 1:100 and cultured in a shaking incubator at 30℃ for 24h. After overnight culture, the bacterial solution was centrifuged at 4000 rpm for 20 min at 4°C, the supernatant was discarded, and the bacterial pellet was resuspended in induction medium BMMY (10 g / L yeast extract, 20 g / L peptone, 0.2 g / L biotin, 13.4 g / L basic nitrogen source medium, 0.5% methanol, 13 mmol / L K2HPO4, 87 mmol / L KH2PO4) to an OD600 value of 1. The induction culture was carried out for 3 days, and 1% methanol was added every 24 h (see the results). Figure 4 A).

[0108] Figure 4 A Results show that: SDS-PAGE analysis of Pichia pastoris expression shows that compared with the original GS115 strain expression supernatant, the GS115 culture supernatant after transformation with the recombinant plasmid has obvious protein expression at 40-50KDa, and the expression of the fusion protein increased significantly after induced expression for 72h. In addition, the molecular weight of the fusion protein expressed by yeast is significantly greater than that of the prokaryotic expression system. The reason for this is that yeast can perform post-translational modification while the prokaryotic expression system does not have this function.

[0109] 2.4 Fusion protein purification and self-assembly

[0110] After 3 days of induction, the culture was centrifuged at 4°C, 4000 rpm for 20 min, the supernatant was centrifuged again at 4°C, 12000 rpm for 20 min, and the supernatant was purified using a Ni affinity column. The purification and self-assembly process was the same as in 2.2. The assembled virus-like particles were also identified by SDS-PAGE, Western Blot, Particle gel and transmission electron microscopy (results are shown in Figure 4 B).

[0111] Figure 4 The results of B show that the purified and dialyzed yeast fusion protein samples were analyzed by transmission electron microscopy ( Figure 4 B), round particles with a diameter of about 50 nm were observed, indicating that yeast fusion proteins can also self-assemble into virus-like particles.

[0112] 3. Mammalian cell expression system

[0113] 3.1 Transfection of ExpiCHO-S suspension cells with recombinant plasmid

[0114] ExpiCHO-S cells were cultured in a shaker at 36.5°C, 8% CO2, 110rpm, and humidity ≥80%. When the cell density reached 4-6×106 / ml and the cells were in good condition, the ExpiCHO expression system kit was used for transfection. The cell density was adjusted to 3-4×106 / ml the day before transfection. The cell density and viability were measured the next day. When the cell density reached 7-10×106 / ml and the viability was ≥95%, transfection was performed. The cell density was diluted to 6×106 / ml with culture medium. 3.2ml ExpiFectamine CHO reagent and 0.8μg plasmid DNA were required for each transfection of 1ml culture. Plasmid DNA and ExpiFectamine CHO reagent were diluted with 1ml culture medium respectively, and then the diluted transfection reagent was added to the diluted plasmid DNA and gently pipetted to mix (Note: the transfection reagent must be added to the plasmid DNA within 5min after dilution, otherwise the transfection performance will be reduced). The transfection reagent and plasmid need to be mixed for 1-5 minutes, and then the complex is slowly added to the cell culture. During the addition, gently rotate the shaker to ensure that the ExpiFectamineDNA CHO complex is evenly distributed in the cell culture. Return the shaker to the shaker and add the enhancer and excipients 18-22 hours after transfection.

[0115] 3.2 Purification of virus-like particles

[0116] On the 7th day after transfection, the cell culture supernatant was collected and centrifuged at 4°C, 4000g for 20min, and the precipitate was discarded; the supernatant was centrifuged again at 4°C, 12000rpm for 20min, and the precipitate was discarded. The cell culture supernatant was concentrated by ammonium sulfate precipitation method, and an equal volume of 4M ammonium sulfate solution was slowly added to the culture supernatant. The supernatant gradually became turbid as the protein precipitated, and was placed at 4°C for 1h to ensure that the protein was fully precipitated. Horizontal centrifugation at 4°C, 4000rpm for 20min, the supernatant was discarded, the precipitate was resuspended and dissolved with 10ml PBS for 20min, and then centrifuged again at 4°C, 12000rpm for 20min to remove the precipitate that was not visible to the naked eye. Then, the virus-like particles were purified by sucrose density gradient centrifugation. The concentrated supernatant was loaded onto a 10% to 60% sucrose gradient and ultracentrifuged at 4°C, 40000g for 4h using a SW28 rotor. The centrifuged samples were sampled from top to bottom in 3 ml layers and the locations of virus-like particles were analyzed by SDS-PAGE, Western Blot, Particle gel and transmission electron microscopy (results see Figure 5 ).

[0117] Figure 5The results show that the WB results of mammalian cell ExpiCHO-S cell expression supernatant purified by sucrose density gradient centrifugation ( Figure 5 A) shows that most of the expression supernatant is unassembled fusion protein monomers and dimers. According to literature reports, samples assembled into virus-like particles should be distributed in layers 7-10. Take the ninth layer of the dialyzed solution, dilute it 100 times, and observe it using a transmission electron microscope ( Figure 5 B), the results show that round particles with a diameter of about 50 nm can be seen, which indicates that mammalian cell fusion proteins can also self-assemble into virus-like particles.

[0118] Example 4

[0119] Immunogenicity identification

[0120] 1 Mouse immunization

[0121] Pathogen-free female BALB / c mice aged 6-8 weeks were selected, and the experimental group was divided into 3 groups, with three mice in each group. Virus-like particles expressed by Escherichia coli, Pichia pastoris, and mammalian cells were mixed with Freund's complete adjuvant in a 1:1 ratio and injected intramuscularly into mice of different groups. The VLP dose was 20 μg / mouse. The samples of mice in the control group were PBS and Freund's complete adjuvant. The booster immunization was performed two weeks after the initial immunization, and blood was collected from the eye sockets of mice one week after the booster immunization. The blood samples were stored at room temperature for 1 hour, and the serum was separated by centrifugation at 4000g for 10 minutes at 4°C.

[0122] 2 Detection of specific antibodies in sera of immune mice

[0123] In order to explore anti-HBc-VLP IgG antibodies containing SARS-CoV-2 spike protein epitope peptides, ELISA was performed on mouse immune serum. The mouse immunized serum was diluted with sterile water, and the diluted serum samples were added to a 96-well plate coated with virus-like particles expressed by different expression systems, incubated at 37°C for 1h, incubated and washed with PBST buffer, and HRP-conjugated anti-mouse IgG was added as a secondary antibody. After incubation and washing, TMB colorimetric solution was added and incubated at 37°C in the dark for 15min, and then the reaction was terminated with 1M sulfuric acid. Finally, the absorbance at 450nm was detected with an enzyme reader, and the immunogenicity of the virus-like particles expressed by the three expression systems was judged by the absorbance values ​​(results are shown in Figure 6 ).

[0124] Figure 6 The results showed that during the experiment, mice injected with recombinant fusion protein virus-like particles did not show abnormal phenomena such as decreased activity and decreased appetite, and their behavior was similar to that of the control group. It was also observed that the weight of the mice increased day by day, indicating that the injection of the antigen did not cause adverse reactions in the mice ( Figure 6A). Seven days after booster immunization, ELISA was used to detect antibodies in mouse serum. The results showed that the experimental group produced specific antibodies against the recombinant fusion protein virus-like particles ( Figure 6 B). When the mouse serum dilution was 1:16000, the absorbance value detected at 450nm of the virus-like particles expressed by the mammalian cell and yeast expression system was still significantly different from that of the negative control group (P<0.01), indicating that the virus-like particles expressed by the mammalian cell and yeast expression system have good immunogenicity and can induce mice to produce high levels of specific antibodies.

[0125] The prior art shows that the mutation sites of the currently popular new coronavirus variants are mainly concentrated in the RBD segment, and these mutations will reduce the protective effect of vaccines or neutralizing antibodies. The tandem epitope peptide virus-like particle vaccine designed in the present invention can produce antibodies against multiple conserved regions outside the RBD domain of the new coronavirus S protein, thereby playing a role in the binding of the virus into the cell, the membrane fusion process, and the cellular immune response. Therefore, the present invention can cope with the continuous mutation of the SARS-CoV-2 virus.

[0126] In summary, serological experiments have proved that the recombinant protein designed by the present invention uses the yeast expression system as an expression vector, and the prepared new coronavirus tandem epitope peptide virus-like particle vaccine can produce relevant antibodies. That is to say, the new coronavirus tandem epitope peptide virus-like particle vaccine provided by the present invention is highly conservative against coronaviruses and new coronavirus mutant strains, has good immunogenicity, and can stimulate the body to produce antigen-specific antibodies; at the same time, the yeast expression system is used as an expression vector, which has the advantages of high yield, low production cost, and high protein purity, and is suitable for large-scale preparation of new coronavirus vaccines.

[0127] The above-described embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A novel coronavirus tandem epitope peptide, characterized in that: The novel coronavirus tandem epitope peptides are composed of the core region epitope peptide S450-500 that binds to ACE2 in the RBD domain, the epitope peptide S556-589 that can induce the production of neutralizing antibodies near the RBD domain and hinder the binding of the RBD domain and ACE2 through steric hindrance, the epitope peptide S624-690 located at the C-terminus of the S1 subunit, which can induce antibodies to prevent membrane fusion by inhibiting host protease-mediated protein hydrolysis, the epitope peptide S809-835 located at the C-terminus of the S2 subunit fusion peptide, which can target and induce immune responses of CD4+T cells and CD8+T cells, and the epitope peptide S1140-1166 located at the C-terminus of the spike protein, which is relatively conserved in coronaviruses, which are linearly connected in sequence.

2. A novel coronavirus tandem epitope peptide as claimed in claim 1, characterized in that: The amino acid sequence of the epitope peptide S450-500 is shown in SEQ ID NO: 3; The amino acid sequence of the epitope peptide S556-589 is shown in SEQ ID NO: 4; The amino acid sequence of the epitope peptide S624-690 is shown in SEQ ID NO: 5; The amino acid sequence of the epitope peptide S809-835 is shown in SEQ ID NO: 6; The amino acid sequence of the epitope peptide S1140-1166 is shown in SEQ ID NO: 7; There is a spacer sequence between any two adjacent epitope peptide segments, and the spacer sequence is GGGGS.

3. A novel coronavirus tandem epitope peptide virus-like particle vaccine, characterized in that: It comprises the core protein of hepatitis B virus and the new coronavirus tandem epitope peptide described in any one of claims 1-2.

4. A novel coronavirus tandem epitope peptide virus-like particle vaccine according to claim 5, characterized in that: There is a spacer sequence between the core protein of the hepatitis B virus and the new coronavirus tandem epitope peptide; the spacer sequence is GGGGS.

5. A novel coronavirus tandem epitope peptide virus-like particle vaccine according to claim 3, characterized in that: The amino acid sequence of the hepatitis B virus core protein and the new coronavirus tandem epitope peptide fusion gene is shown in SEQ ID NO:

1.

6. A nucleic acid, characterized in that The active ingredient encoding the new coronavirus tandem epitope peptide virus-like particle vaccine according to any one of claims 3-4, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

2.

7. A method for preparing a novel coronavirus tandem epitope peptide virus-like particle vaccine according to any one of claims 5-6, characterized in that: include: Use flexible linker 3 to insert the novel coronavirus tandem epitope peptide of any one of claims 1-2 into the immunodominant epitope of HBc, i.e., between positions 79-81 of the amino acid sequence, to obtain a recombinant plasmid, and then purify the recombinant plasmid through an expression system to obtain a novel coronavirus tandem epitope peptide virus-like particle vaccine.

8. The method for preparing a novel coronavirus tandem epitope peptide virus-like particle vaccine according to claim 7, characterized in that: The expression system is any one of a yeast expression system and a mammalian expression system.

9. A new coronavirus tandem epitope peptide virus-like particle vaccine prepared by the preparation method of a new coronavirus tandem epitope peptide virus-like particle vaccine according to claim 8.

10. Use of the novel coronavirus tandem epitope peptide virus-like particle vaccine described in claim 5, 6 or 9 in the preparation of the novel coronavirus tandem epitope peptide virus-like particle vaccine.

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